Method of binding water-soluble proteins and water-soluble peptides to water-insoluble polymers using cyanogen halide

ABSTRACT

Cyanogen halides are employed for binding water-soluble proteins and water-soluble peptides containing a primary or secondary amino group to a water-insoluble polymer containing one or more hydroxyl and/or primary and/or secondary amino groups. The invention is useful, among other things, for binding watersoluble enzymes to water-insoluble polymers while preserving the activity of the enzyme.

iJiied ties arena Amen et a1. Feb. 29, 1972 [54] METHOD OF BINDING WATE-3,167,485 l/l965 Katchalski et aI. ..l95/63 SQLUBLE EH S B WATER,3,278,392 10/1966 Patchornik ..195/63 Ofstead X ENSULULE PULYMERS USINGFOREIGN PATENTS OR APPLICATIONS QYANQGEN HAMDE 84,196 1/1958 Denmark[72] Inventors: Rolf E. A. V. Axen, Simonsbo, Upplands ,96 965 reatBritain Balinge; Jerlcer Olaf Porath, Karlsrogatan 42; Erik SverkerEmbacli, Vfltgatan 8, OTHER PUBLICATIONS both of Uppsala, all of Sweden1 Nature, Vol. 188, I960 Bar-Eli et al., pp. 856-857 [73] Assignee.2213622112203 AB, B orkgatan 30, Uppsala, mmy q h m g ly q 2 pp 2 8 dgWeliky etal. 1965 [22] Filed: Mar. 6, 1970 Ann. Rev. of Biochemistry,Vol. 35, part II, 1966. pp. 873- 880 and 896-901, Silman et al. Nature,214mm, q 1t l-, P91202994,

Related US. Application Data Prima ExaminerI-Ioward E. Schain [63]iCggstmzligislzrgrilr-gart of Ser. No. 729,914, May 17, g, C. philpm[30] Foreign Application Priority Data [57] ABSTRACT May 23,1967 Sweden..7I69/67 y g halides are p y for binding watepsoluble [52] U S C]195/68 195/63 195 lDlG 11 proteins and water-soluble peptides containinga primary or R 260;l12 7 424/} 42/12 secondary amino group to awater-insoluble polymer contain- 424/94 ing one or more hydroxyl and/orprimary and/or secondary [51] Im CH C(Wg 7/00 CO-Ig 7/02 amino groups.The invention is useful, among other things, for [58] a /63 68 260/112binding water-soluble enzymes to water-insoluble polymers h V whilepreserving the activity of the enzyme. [56] Reierences Cited 8 Claims,No Drawings METHOD OF BlNDlNG WATER-SOLUBLE PROTEINS AND WATER-SOLUBLEPEPTIDES T WATER- INSOLUBLE POLYMERS USING CYANOGEN HALHDE Thisapplication is a continuation-in-part of Ser. No. 729,914 filed May 17,1968 and now abandoned.

The present invention concerns a method of binding, by covalent bonds,water-soluble proteins and water-soluble peptides containing one or moregroups of the formula -Yl-l, in which YH represents a primary orsecondary amino group, to water-insoluble polymers containing one ormore groups of the formula --XH, in which Xl-l represents a hydroxylgroup or a primary or secondary amino group. The group Xl-l as well asthe group -Yl-l contains a reactive hydrogen atom H.

The invention is characterized in that the water-insoluble polymercontaining one or more groups of the formula -XH is reacted with acyanogen halide and with the water'soluble protein or water-solublepeptide. The reaction is carried out in an aqueous weakly alkalinemedium. lt is thus carried out in the presence of water, which is ofspecial significance; and the temperature is variable, for example, inthe range of from 0 to 50 C. The reaction is based on the formation ofbridges with covalent bonds between the water-insoluble polymer and thewater-soluble protein or peptide. Investigations carried out haverevealed that the bridges have the formula A Q-... ..l H

Z... wherein A is the residue of the water-insoluble polymer and B isthe residue of the protein or peptide and Z stands for imino =Nl-l) oroxygen (=0). That Z can also stand for oxygen is due to the fact thatthe imino group can in certain cases convert into oxygen by hydrolysis.

According to the invention, the reaction can be carried out in one ormore stages. As a rule it is carried out, however, in two stages. Thefirst of these stages is commonly effected in such a manner that thewater-insoluble polymer containing one or more groups having the formulaXH is contacted with a cyanogen halide, the latter being present in theform of the pure substance or a solution thereof. As a cyanogen halide,there may usually be used the iodo-, chloro, or bromocompound oroptionally a mixture thereof. The reaction is carried out under alkalineconditions, which are provided by the addition of a suitable alkalinereacting substance to the aqueous reaction medium, such as sodiumhydroxide. Suitable pH values are primarily such values in the range offrom 8 to 13. If XH is a primary or secondary amino group, it ispossible to work in a more weakly alkaline medium than when -XH is ahydroxy group. The skilled person can by simple preliminary testsreadily determine the suitable pH value or pH interval. The reaction canbe carried out at different temperatures, for example, in the range offrom 0 to 50 C. When working in aqueous solution, certain losses ofcyanogen halide arises due to part of the latter being consumed byhydrolysis. The reaction results in the formation of reactivederivatives of the water-insoluble polymer which can then immediately oroptionally after isolation, be further reacted with the water-solubleprotein or water-soluble peptide. The reactive derivative as formed canalso be stored under suitable conditions and later be reacted with thebiopolymer. Prior to being bound to the biopolymer, a reactivederivative based on a water-insoluble polymer can be purified, forexample by washing.

The second stage of the process is preferably carried out under weaklyalkaline conditions at a temperature which can, for example, be in therange of from 0 to 50 C., such as at room temperature. it is anadvantage of the invention that this stage can be carried out in thepresence of water.

An important advantage of the process of the invention is that sensitivewater-soluble proteins or water-soluble peptides containing one or moregroups -YH can be bound to waterinsoluble polymers of the kind set forthin the preamble, without the proteins or peptides being destroyed orundergoing any other undesired change. It is possible to bind theproteins and the peptides without the peptide linkages being broken up.According to the invention it is thus, for example, possible to bindwater-soluble enzymes to water-insoluble polymers essentially withoutlosses in enzyme activity and to bind an antibody in solution to theinsoluble polymer, without loosing the ability of the antibody to bindits antigen. The process according to the invention can thus be said torepresent a surprisingly mild and simple method of binding thewater-soluble protein or water-soluble peptide to water-insolublepolymers of the kind given above and represents a very valuable methodof insolubilizing water-soluble proteins and peptides.

According to the invention, the water-insoluble polymer can be awater-insoluble polysaccharide or a water-insoluble derivative of apolysaccharide which contains hydroxyl groups and/or primary and/orsecondary amino groups. An example of a water-insoluble polysaccharideis cellulose. Examples of insoluble polysaccharide derivatives arecross-linked polysaccharides, for example, cross-linked dextran, starch,dextrin, and hydroxyl group-containing derivatives of suchpolysaccharides such as hydroxyethyl group-containing derivatives oramino group-containing derivatives such as paminophenoxyhydroxypropylgroup-substituted cross-linked dextran and starch. Another example isagarose. A still further example is water-insoluble cross-linkedpolyvinyl-alcohol and synthetic polymers containing amino groups.

The average molecular weight of the selected polymer, containing one ormore groups XH, can vary within very broad limits. Preferably it should,however, by more than about 1 ,000, for example, more than about 10,000.

According to the invention, the polymer is insoluble in water, but itcan be swellable in water. In this connection, it can consist of across-linked three-dimensional hydroxyl group-containing network whichis insoluble but swellable in water. As examples of such polymers may bementioned copolymers of dextran with a bifunctional substance, such asepichlorohydrin, such copolymers being characterized by presenting avarying ability of absorbing water. Copolymers of other polysaccharidesor other hydroxyl compounds with bifunctional substances may come inquestion, such as copolymers of saccharose and of sugar alcohols, suchas sorbitol, mannitol, and polyvinyl-alcohol. As copolymers may also bementioned those which are substituted by primary or secondary aminogroups such as p-aminophenoxyhydroxypropyl groups.

As water-soluble proteins and peptides, containing one or more groups ofthe formula --Yl-l, which are to be subjected to the binding reactionwith the reactive derivative according to the invention may be mentionedenzymes, antibodies, protein and/or peptide hormones or antigenicproteins. The products according to the invention are of great interest.An enzyme which is bound to a water-insoluble polymer according to theinvention can thus be packed into a bed and this bed be utilized as areactor for chemical reactions. In this connection, a solution of asubstrate can be passed through the enzyme bed to convert this substrateinto a valuable product. The reaction is automatically stopped when thesolution leaves the bed. The solid enzyme can be used for a long time ina continuous operation.

Another valuable example is antibodies bound to water-insoluble polymersaccording to the present invention. Such products can be combinedspecifically with the corresponding antigen, for example, for theanalytical determination of the latter.

EXAMPLE l Binding of chymotrypsin to a copolymer of dextran withepichlorohydrin A. Activation of the copolymer by reacting with cyanogenbromide Two hundred milligrams copolymer of dextran with epichlorohydrin(SEPl-lADEX (3-200 from Pharmacia Fine Chemicals AB, Uppsala, Sweden) awater-insoluble gel product which is swellable in water were combinedwith 8 ml. of an aqueous solution containing 25 mg. of cyanogen bromideper ml. of water. The reaction was allowed to take place at 23 C. whilestirring and the pH of the mixture was maintained at 11.5 using anautomatic titrator adding 2 M sodium hydroxide solution. The reactiontime was 6 minutes. The product was washed rapidly with suction on aglass filter with cold water, and used for the next stage of thereaction.

B. Binding with chymotrypsin The activated copolymer obtained accordingto (A) was contacted with 200 mg. of chymotrypsin in 2 ml. of 0.1 Msodium hydrocarbonate solution, whereupon the mixture was allowed toreact at 23 for 30 hours while being stirred. Sub sequent to beingsubjected to suction on a glass filter the obtained product was washedwith 0.1 M sodium hydrocarbonate solution, 10 M hydrochloric acid,water, 0.5 M sodium chloride solution, water and a water-acetone mixturewith increasing concentrations of acetone, and finally with pureacetone. The shrunken product was then dried. The result obtained uponanalysis of amino acids was a content of 200 mg. of protein per gram ofdried final product. The product obtained has enzymatic activity and isused, for example, for analytical procedures.

EXAMPLE 2 Binding of insulin to a copolymer of dextran withepichlorohydrin A. Activation of the copolymer by reacting with cyanogeniodine.

Fifty milligrams of the water-insoluble copolymer disclosed in Example 1were contacted with 2 ml. of an aqueous solution containing 30 mg. ofcyanogen iodide per ml. of water. The reaction took place at 23 C. whilestirring, the pH of the mixture being 1 l and the reaction period 25minutes. In other respects the sequence of steps was identical to thatin Example B. Binding with insulin The activated copolymer obtainedaccording to A was added with 0.5 M sodium hydrocarbonate solution andthe residue removed from the gel by suction. This was then contactedwith 11.5 mg. of dezincificated insulin (from swine) in 150 pl. of 0.5 Msodium hydrocarbonate solution. The reaction took place for 5 hours at23 C. The product was washed and dried in a manner similar to thatdescribed in Example 1. The result obtained upon analysis of amino acidswas a binding yield of 24 mg. of insulin per gram of dried reactionproduct. The insoluble product is able to bind antibodies againstinsulin. The product can be used for analytical procedures.

EXAMPLE 3 Binding of 'y-globulin (antichorionic gonadotropin) to aminogroup-substituted (p-aminophenoxyhydroxypropylether) copolymer ofdextran with epichlorohydrin A. Activation ofp-aminophenoxyhydroxypropylether of copolymerisate One hundredmilligrams of p-arninophenoxyhydroxypropylether of the water-insolublecross-linked dextran containing approximately 250 prnol of amino groupsper gram of amino polymer were swollen in 3 ml. of water. Fiftymilligrams of cyanogen bromide were dissolved in 3 ml. of water andadded to the gel while stirring; the H was adjusted to 8 and maintainedat this level for 5 minutes by adding 0.5 M of sodium hydroxide.Subsequent to the termination of the reaction the gel was washed rapidlywith cold water and 0.1 M sodium bicarbonate solution, and was thenimmediately used for the binding process.

B. Binding with y-globulin (antichorionic gonadotropin) Antiserumagainst human chorionic gonadotropin was ob tained from rabbits afterinjection of the hormones in Freunds adjuvant. The immunoglobulins wereprecipitated by adding half the volume of saturated ammonium sulphatesolution at room temperature, which was repeated twice. Thelast-obtained precipitate was dissolved in and diaiyzed against 0.l Msodium bicarbonate solution.

0.5 ml. of antichorionic gonadotropin solution corresponding to 0.5 ml.of antiserum were reacted with the activated polymer for 5 hours at 23C. The product was washed with 0.1 M of sodium bicarbonate solution, 0.5M of sodium chloride solution, water, 10" M of hydrochloric acid andwater. A portion of the product was shrunken and dried for analysis andwas found to contain about 3.5 mg. of protein per gram of polymer. Thebound antibodies are capable of binding the corresponding antigen(chorionic gonadotropin).

The majority of the product was used for radioimmunologicaldeterminations of chorionic gonadotropin. The product showed splendidproperties for binding chorionic gonadotropin and can be used foranalytical procedures.

EXAMPLE 4 Binding of oxytocin to cellulose A. Activation of cellulose byreacting with cyanogen bromide Cellulose (Munktell Powder No. 400) wasmercerized by treatment with 17.5 percent sodium hydroxide solution at 0C. for 24 hours and in a nitrogen gas atmosphere. Fifty milligrams ofmercerized cellulose was activated with 2 ml. of a cyanogen bromidesolution containing 25 mg. of cyanogen bromide per ml. of water at apl-l of 11 for 12 minutes at C. The product was washed with cold waterand added with 0.1 M of a sodium hydrocarbonate solution; the residuebeing removed by suction.

B. Binding with oxytocin The activated cellulose was reacted with 10 mg.of oxytocin in 1 ml. of 0.5 M sodium hydrocarbonate solution for 10hours at 23 C. The obtained product was washed and shrunken as describedin Example 1(B). Analysis resulted in a binding yield of mg. of oxytocinper gram of dried reaction product.

EXAMPLE 5 Binding of glucose oxidase to agaros A. Activation of agarosby reaction with cyanogen bromide Water-swollen, ball-shaped agarosparticles (Sepharose 213) were subjected to suction on a glass filter.3.9 g. (correspond ing to mg. of shrunken and dried agaros) were addedwith 4 ml. of a cyanogen bromide solution containing 25 mg. cyanogenbromide per ml. of water, whereafter the activation process was efiectedanalogously with Example 1(8) at pH 11 by adding 2 M sodium hydroxidesolution for 6 minutes using an automatic titrator at 23 C. Theactivated product was washed on a glass filter with l l. of ice waterand was finally washed rapidly with 0.1 M phosphate buffer having a pHof 7.4.

B. Binding with glucose oxidase The activated polymer was reacted with24 mg. of glucose oxidase (purified from Worthington Biochemical Corp.)which was dissolved in 1 ml. of 0.1 M phosphate buffer at pH 7.4. Thebinding process took place for 10 hours with slow rotation of thereaction vessel. The gel conjugate was placed in a column and washedwith 1 liter of 0.1 M acetate buffer of pill 4.6, this amount beingpumped by means of a pump through the column for 10 hours.

A portion of the gel was removed, shrunken with acetone, dried andanalyzed with respect to protein. The amount of bonded glucose oxidasewas calculated therefrom to reach 30 mg. per g. of dried polymerconjugate.

The bound enzyme was found to have considerable activity, and thecatalytic column could be used for determining glucose in samples. Thecolumn was also found to possess good chromatographic properties. Thewater-insoluble product is a valuable reagent for analytical procedures.

EXAMPLE 6 Binding of chymotrypsin to polyacrylamide polymer substitutedby aromatic amino groups A. Activation of polymer by reacting withcyanogen bromide Cross-linked polyacrylamide polymer substituted bypaminophenyl groups, the major structural features of which polymer areevident from the following formula was used (Enzacryl; a productmarketed by The United Kingdom company Koch-Light Laboratories Ltd.,Colmbrook, Buckingham Shire) o ONHz c ONHz ONE.

NH-CO r q H: c ONH@NH2 I\IIH-CO L N112 coma in order to activate thepolymer, 100 mg. thereof were added with 4 ml. of a cyanogen bromidesolution containing mg. of cyanogen bromide per ml. of water, whereafterthe activation process was effected at pH 9.0 for 6 minutes at roomtemperature. The activated gel was washed on a glass filter with suctionfor 5 min. with 0.1 M sodium bicarbonate solution.

B. Binding with chymotrypsin The activated gel was suspended in 2 ml.0.1 M sodium bicarbonate solution and 25 mg. chymotrypsin was added. Thecoupling was allowed to proceed at 4 and under slow stirring for 16hours.

The resulting product was washed at room temperature in a small column.The washing solutions were supplied at mL/h. by a peristaltic pump. Thefollowing washing solutions were used in order: 0.1 M sodium bicarbonatesolution (24 hours); l0 M hydrochloric acid (1 hour); 0.5 M sodiumchloride solution (24 hours); distilled water (3 hours); 0.01 M sodiumacetate buffer of pH 5.4 (1 hour). The product can be stored as asuspension in the last-mentioned buffer.

The enzymatic activity of the conjugate was determined toward a l4-mMsolution of N-acetyl-L-tyrosine ethyl ester in an aqueous mediumcontaining 5 percent of ethanol. The substrate was hydrolyzed at a rateof 3.5 pmol per min per mg. of dried conjugate. The content of proteinwas I05 mg. per g. of dried conjugate. The activity of the fixed enzymewas therefore about 35 pmol per min. per mg. The activity of the freechymotrypsin was umol per min. per mg.

What we claim is:

1. In the method for binding, by covalent bonds a. a water-insolublepolymer containing at least one group of the formula -XH, wherein --XHrepresents a member selected from the group consisting of hydroxylgroups, primary and secondary amino groups, and

b. a member selected from the group consisting of watersoluble proteinsand water-soluble peptides containing at least one group of the formula-YH wherein YH represents a member selected from the group consisting ofprimary and secondary amino groups, the improvement which comprises:

1. first causing said water-insoluble polymer of (a) to react with acyanogen halide in an aqueous alkaline medium,

2. thereafter reacting the water-insoluble polymer ob tained from step(1) with a member selected from the group consisting of water-solubleproteins and watersoluble peptides of the type specified in (b).

2. The method according to claim 1, wherein said water-insoluble polymeris selected from the group consisting of waterinsoluble polysaccharidesand water-insoluble polysaccharide derivatives containing a memberselected from the group consisting of hydroxyl groups and primary andsecondary amino groups.

3. The method as set forth in claim 1, wherein said water-insolublepolymer is insoluble but swellable in water.

4. The method as claimed in claim 1, wherein said water-insolublepolymer is a cross-linked three-dimensional hydroxyl group-containingnetwork.

5. The method as claimed in claim 1, wherein the polymer is a copolymerof dextran with epichlorohydrin.

6. The method as set forth in claim 1, wherein the watersoluble peptideis an enzyme.

7. The method as set forth in claim 1, wherein the member selected fromthe group consisting of water-soluble proteins and water-solublepeptides is an antibody.

8. The method as claimed in claim 1, wherein the member selected fromthe group consisting of water-soluble proteins and water-solublepeptides is an antigen.

2. thereafter reacting the water-insoluble polymer obtained from step(1) with a member selected from the group consisting of water-solubleproteins and water-soluble peptides of the type specified in (b).
 2. Themethod according to claim 1, wherein said water-insoluble polymer isselected from the group consisting of water-insoluble polysaccharidesand water-insoluble polysaccharide derivatives containing a memberselected from the group consisting of hydroxyl groups and primary andsecondary amino groups.
 3. The method as set forth in claim 1, whereinsaid water-insoluble polymer is insoluble but swellable in water.
 4. Themethod as claimed in claim 1, wherein said water-insoluble polymer is across-linked three-dimensional hydroxyl group-containing network.
 5. Themethod as claimed in claim 1, wherein the polymer is a copolymer ofdextran with epichlorohydrin.
 6. The method as set forth in claim 1,wherein the water-soluble peptide is an enzyme.
 7. The method as setforth in claim 1, wherein the member selected from the group consistingof water-soluble proteins and water-soluble peptides is an antibody. 8.The method as claimed in claim 1, wherein the member selected from thegroup consisting of water-soluble proteins and water-soluble peptides isan antigen.